Restraining of glycoprotein VI- and integrin α2ß1-dependent thrombus formation by platelet PECAM1.

The platelet receptors, glycoprotein VI (GPVI) and integrin α2ß1 jointly control collagen-dependent thrombus formation via protein tyrosine kinases. It is unresolved to which extent the ITIM (immunoreceptor tyrosine-based inhibitory motif) receptor PECAM1 and its downstream acting protein tyrosine phosphatase PTPN11 interfere in this process. Here, we hypothesized that integrin α2ß1 has a co-regulatory role in the PECAM1- and PTPN11-dependent restraint of thrombus formation. We investigated platelet activation under flow on collagens with a different GPVI dependency and using integrin α2ß1 blockage. Blood was obtained from healthy subjects and from patients with Noonan syndrome with a gain-of-function mutation of PTPN11 and variable bleeding phenotype. On collagens with decreasing GPVI activity (types I, III, IV), the surface-dependent inhibition of PECAM1 did not alter thrombus parameters using control blood. Blockage of α2ß1 generally reduced thrombus parameters, most effectively on collagen IV. Strikingly, simultaneous inhibition of PECAM1 and α2ß1 led to a restoration of thrombus formation, indicating that the suppressing signaling effect of PECAM1 is masked by the platelet-adhesive receptor α2ß1. Blood from 4 out of 6 Noonan patients showed subnormal thrombus formation on collagen IV. In these patients, effects of α2ß1 blockage were counterbalanced by PECAM1 inhibition to a normal phenotype. In summary, we conclude that the suppression of GPVI-dependent thrombus formation by either PECAM1 or a gain-of-function of PTPN11 can be overruled by α2ß1 engagement.

Porcine-derived collagen peptides promote re-epithelialisation through activation of integrin signalling.

Chronic non-healing cutaneous wounds represent a major burden to patients and healthcare providers worldwide, emphasising the continued unmet need for credible and efficacious therapeutic approaches for wound healing. We have recently shown the potential for collagen peptides to promote proliferation and migration during cutaneous wound healing. In the present study, we demonstrate that the application of porcine-derived collagen peptides significantly increases keratinocyte and dermal fibroblast expression of integrin α2ß1 and activation of an extracellular signal-related kinase (ERK)-focal adhesion kinase (FAK) signalling cascade during wound closure in vitro. SiRNA-mediated knockdown of integrin ß1 impaired porcine-derived collagen peptide-induced wound closure and activation of ERK-FAK signalling in keratinocytes but did not impair ERK or FAK signalling in dermal fibroblasts, implying the activation of differing downstream signalling pathways. Studies in ex vivo human 3D skin equivalents subjected to punch biopsy-induced wounding confirmed the ability of porcine-derived collagen peptides to promote wound closure by enhancing re-epithelialisation. Collectively, these data highlight the translational and clinical potential for porcine-derived collagen peptides as a viable therapeutic approach to promote re-epithelialisation of superficial cutaneous wounds.

Functional heterogeneity of IFN-γ-licensed mesenchymal stromal cell immunosuppressive capacity on biomaterials.

Mesenchymal stromal cells (MSCs) are increasingly combined with biomaterials to enhance their therapeutic properties, including their immunosuppressive function. However, clinical trials utilizing MSCs with or without biomaterials have shown limited success, potentially due to their functional heterogeneity across different donors and among different subpopulations of cells. Here, we evaluated the immunosuppressive capacity, as measured by the ability to reduce T-cell proliferation and activation, of interferon-gamma (IFN-γ)-licensed MSCs from multiple donors on fibrin and collagen hydrogels, the two most commonly utilized biomaterials in combination with MSCs in clinical trials worldwide according to ClinicalTrials.gov Variations in the immunosuppressive capacity between IFN-γ-licensed MSC donors on the biomaterials correlated with the magnitude of indoleamine-2,3-dioxygenase activity. Immunosuppressive capacity of the IFN-γ-licensed MSCs depended on the αV/α5 integrins when cultured on fibrin and on the α2/ß1 integrins when cultured on collagen. While all tested MSCs were nearly 100% positive for these integrins, sorted MSCs that expressed higher levels of αV/α5 integrins demonstrated greater immunosuppressive capacity with IFN-γ licensing than MSCs that expressed lower levels of these integrins on fibrin. These findings were equivalent for MSCs sorted based on the α2/ß1 integrins on collagen. These results demonstrate the importance of integrin engagement to IFN-γ licensed MSC immunosuppressive capacity and that IFN-γ-licensed MSC subpopulations of varying immunosuppressive capacity can be identified by the magnitude of integrin expression specific to each biomaterial.

Two-Dimensional Peptide Assembly via Arene-Perfluoroarene Interactions for Proliferation and Differentiation of Myoblasts.

Supramolecular assembly based on aromatic interactions can provide well-defined nanostructures with an understanding of intermolecular interactions at the molecular level. The peptide assembly via a supramolecular approach can overcome the inherent limitations of bioactive peptides, such as proteolytic degradations and rapid internalizations into the cytosol. Although extensive research has been carried out on supramolecular peptide materials with a two-dimensional (2D) structure, more needs to be reported on biological activity studies using well-defined 2D peptide materials. Physical and chemical properties of the 2D peptide assembly attributed to their large surface area and flexibility can show low cytotoxicity, enhanced molecular loading, and higher bioconjugation efficiency in biological applications. Here, we report supramolecular 2D materials based on the pyrene-grafted amphiphilic peptide, which contains a peptide sequence (Asp-Gly-Glu-Ala; DGEA) that is reported to bind to the integrin α2ß1 receptor in 2D cell membranes. The addition of octafluoronaphthalene (OFN) to the pyrene-grafted peptide could induce a well-ordered 2D assembly by face-centered arene-perfluoroarene stacking. The DGEA-peptide 2D assembly with a flat structure, structural stability against enzymatic degradations, and a larger size can enhance the proliferation and differentiation of muscle cells via continuous interactions with cell membrane receptors integrin α2ß1 showing a low intracellular uptake (15%) compared to that (62%) of the vesicular peptide assembly. These supramolecular approaches via the arene-perfluoroarene interaction provide a strategy to fabricate well-defined 2D peptide materials with an understanding of assembly at the molecular level for the next-generation peptide materials.

Glomerular basement membrane deposition of collagen α1(III) in Alport glomeruli by mesangial filopodia injures podocytes via aberrant signaling through DDR1 and integrin α2ß1.

In Alport mice, activation of the endothelin A receptor (ETA R) in mesangial cells results in sub-endothelial invasion of glomerular capillaries by mesangial filopodia. Filopodia deposit mesangial matrix in the glomerular basement membrane (GBM), including laminin 211 which activates NF-κB, resulting in induction of inflammatory cytokines. Herein we show that collagen α1(III) is also deposited in the GBM. Collagen α1(III) localized to the mesangium in wild-type mice and was found in both the mesangium and the GBM in Alport mice. We show that collagen α1(III) activates discoidin domain receptor family, member 1 (DDR1) receptors both in vitro and in vivo. To elucidate whether collagen α1(III) might cause podocyte injury, cultured murine Alport podocytes were overlaid with recombinant collagen α1(III), or not, for 24 h and RNA was analyzed by RNA sequencing (RNA-seq). These same cells were subjected to siRNA knockdown for integrin α2 or DDR1 and the RNA was analyzed by RNA-seq. Results were validated in vivo using RNA-seq from RNA isolated from wild-type and Alport mouse glomeruli. Numerous genes associated with podocyte injury were up- or down-regulated in both Alport glomeruli and cultured podocytes treated with collagen α1(III), 18 of which have been associated previously with podocyte injury or glomerulonephritis. The data indicate α2ß1 integrin/DDR1 co-receptor signaling as the dominant regulatory mechanism. This may explain earlier studies where deletion of either DDR1 or α2ß1 integrin in Alport mice ameliorates renal pathology. © 2022 Boys Town National Research Hospital. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Melatonin suppresses the metastatic potential of osteoblastic prostate cancers by inhibiting integrin α2 ß1 expression.

Advanced prostate cancer often develops into bone metastasis, which is characterized by aberrant bone formation with chronic pain and lower chances of survival. No treatment exists as yet for osteoblastic bone metastasis in prostate cancer. The indolamine melatonin (N-acetyl-5-methoxytryptamine) is a major regulator of the circadian rhythm. Melatonin has shown antiproliferative and antimetastatic activities but has not yet been shown to be active in osteoblastic bone lesions of prostate cancer. Our study investigations reveal that melatonin concentration-dependently decreases the migratory and invasive abilities of two osteoblastic prostate cancer cell lines by inhibiting FAK, c-Src, and NF-κB transcriptional activity via the melatonin MT1 receptor, which effectively inhibits integrin α2 ß1 expression. Melatonin therapy appears to offer therapeutic possibilities for reducing osteoblastic bone lesions in prostate cancer.

Development and preliminary evaluation of an integrin α2ß1-targeted PET probe as a supplement and alternative of PSMA imaging for prostate cancer.

An integrin α2ß1-targeted PET probe (68Ga-IABtP) was developed to serve as a supplement and alternative of PSMA imaging for prostate cancer. 68Ga-IABtP was synthesized by labeling the precursor peptide with 68Ga with 93% labeling yield and 4.14 MBq/µg specific radioactivity. 68Ga-IABtP showed no specific uptake in LNCaP prostate cancer cell with low integrin α2ß1 expression but significantly increased uptake in PC-3 prostate cancer cell with high integrin α2ß1 expression, which could be specifically blocked by the integrin α2ß1 monoclonal antibody. The efflux experiments demonstrated that 68Ga-IABtP could rapidly penetrate into PC-3 cell after cell binding, thereby prolonging the residence time in the tumor and allow enough time for probe clearance from the circulation and non-specific organs. The biodistribution study indicated that 68Ga-IABtP showed no specific accumulation in non-target organs and was quickly cleared from the kidney. The in vivo PET-CT imaging demonstrated that 68Ga-IABtP showed no specific uptake in LNCaP tumor but could specifically accumulate in the PC-3 tumor, and was rapidly cleared from spleen, intestine, kidney and liver, resulting in excellent contrast effect with low background signal and high target to non-target ratios.

α2ß1 Integrin Is Required for Optimal NK Cell Proliferation during Viral Infection but Not for Acquisition of Effector Functions or NK Cell-Mediated Virus Control.

NK cells play an important role in antiviral resistance. The integrin α2, which dimerizes with integrin ß1, distinguishes NK cells from innate lymphoid cells 1 and other leukocytes. Despite its use as an NK cell marker, little is known about the role of α2ß1 in NK cell biology. In this study, we show that in mice α2ß1 deficiency does not alter the balance of NK cell/ innate lymphoid cell 1 generation and slightly decreases the number of NK cells in the bone marrow and spleen without affecting NK cell maturation. NK cells deficient in α2ß1 had no impairment at entering or distributing within the draining lymph node of ectromelia virus (ECTV)-infected mice or at becoming effectors but proliferated poorly in response to ECTV and did not increase in numbers following infection with mouse CMV (MCMV). Still, α2ß1-deficient NK cells efficiently protected from lethal mousepox and controlled MCMV titers in the spleen. Thus, α2ß1 is required for optimal NK cell proliferation but is dispensable for protection against ECTV and MCMV, two well-established models of viral infection in which NK cells are known to be important.

The voltage-gated K+ channel Kv1.3 modulates platelet motility and α2ß1 integrin-dependent adhesion to collagen.

Kv1.3 is a voltage-gated K+-selective channel with roles in immunity, insulin-sensitivity, neuronal excitability and olfaction. Despite being one of the largest ionic conductances of the platelet surface membrane, its contribution to platelet function is poorly understood. Here we show that Kv1.3-deficient platelets display enhanced ADP-evoked platelet aggregation and secretion, and an increased surface expression of platelet integrin αIIb. In contrast, platelet adhesion and thrombus formation in vitro under arterial shear conditions on surfaces coated with collagen were reduced for samples from Kv1.3-/- compared to wild type mice. Use of collagen-mimetic peptides revealed a specific defect in the engagement with α2ß1. Kv1.3-/- platelets developed significantly fewer, and shorter, filopodia than wild type platelets during adhesion to collagen fibrils. Kv1.3-/- mice displayed no significant difference in thrombus formation within cremaster muscle arterioles using a laser-induced injury model, thus other pro-thrombotic pathways compensate in vivo for the adhesion defect observed in vitro. This may include the increased platelet counts of Kv1.3-/- mice, due in part to a prolonged lifespan. The ability of Kv1.3 to modulate integrin-dependent platelet adhesion has important implications for understanding its contribution to normal physiological platelet function in addition to its reported roles in auto-immune diseases and thromboinflammatory models of stroke.

Collagen Type XI Inhibits Lung Cancer-Associated Fibroblast Functions and Restrains the Integrin Binding Site Availability on Collagen Type I Matrix.

The tumor microenvironment, including cancer-associated fibroblast (CAF), plays an active role in non-small cell lung cancer (NSCLC) development and progression. We previously reported that collagen type XI and integrin α11, a collagen receptor, were upregulated in NSCLC; the latter promotes tumor growth and metastasis. We here explored the role of collagen type XI in NSCLC stroma. We showed that the presence of collagen type XI in collagen type I matrices inhibits CAF-mediated collagen remodeling and cell migration. This resulted in the inhibition of CAF-dependent lung-tumor cell invasion. Among the collagen receptors expressed on CAF, we determined that DDR2 and integrin α2ß1, but not integrin α11ß1, mediated the high-affinity binding to collagen type XI. We further demonstrated that collagen type XI restrained the integrin binding site availability on collagen type I matrices, thus limiting cell interaction with collagen type I. As a consequence, CAFs failed to activate FAK, p38 and Akt one hour after they interacted with collagen type I/XI. We concluded that collagen type XI may have a competitive negative feedback role on the binding of collagen type I to its receptors.

Integrin α2ß1 inhibition attenuates prostate cancer cell proliferation by cell cycle arrest, promoting apoptosis and reducing epithelial-mesenchymal transition.

Integrin α2ß1 plays an important role in cellular migration and metastasis processes associated with prostate cancer. The aim of this study was to assess whether selective inhibition of integrin α2ß1 is an effective strategy to target metastatic prostate cancer cells. In this regard, we examined the effects of the inhibitor BTT-3033, which selectively interferes with the connection between integrin a2b1 and its ligand, on migration, epithelial-mesenchymal transition (EMT), cell cycle arrest, apoptosis, and specific intracellular signaling pathways using LNcap-FGC and DU-145 prostate cancer cell lines. Western blot analysis and immunocytochemistry assays showed that inhibition of integrin a2b1 inhibits EMT, through the increased expression of E-cadherin and decreased expression of N-cadherin and vimentin. Scratch wound healing assays revealed a direct effect on integrin α2ß1 in the migration capacity of cells. In addition, treatment with BTT-3033 induced a reduction in cell viability and proliferation, as assessed by MTT and BrdU assays. In addition, the results show that BTT-3033 inhibits cell proliferation by inducing G1 cell cycle arrest. Moreover, inhibition of integrin α2ß1 induces apoptosis through the activation of ROS, Bax protein upregulation, caspase-3 activation, and depletion of ΔΨm.  Molecular signaling studies showed that integrin α2ß1 was a positive regulator of MKK7 phosphorylation. In conclusion, our results reveal a critical role for integrin a2b1 in the proliferation of prostate cancer cells, as demonstrated by EMT inhibition, cell cycle arrest, and apoptosis induction in response to treatment with its specific inhibitor BT-3033.

Selectivity of the collagen-binding integrin inhibitors, TC-I-15 and obtustatin.

Integrins are a family of 24 adhesion receptors which are both widely-expressed and important in many pathophysiological cellular processes, from embryonic development to cancer metastasis. Hence, integrin inhibitors are valuable research tools which may have promising therapeutic uses. Here, we focus on the four collagen-binding integrins α1ß1, α2ß1, α10ß1 and α11ß1. TC-I-15 is a small molecule inhibitor of α2ß1 that inhibits platelet adhesion to collagen and thrombus deposition, and obtustatin is an α1ß1-specific disintegrin that inhibits angiogenesis. Both inhibitors were applied in cellular adhesion studies, using synthetic collagen peptide coatings with selective affinity for the different collagen-binding integrins and testing the adhesion of C2C12 cells transfected with each. Obtustatin was found to be specific for α1ß1, as described, whereas TC-I-15 is shown to be non-specific, since it inhibits both α1ß1 and α11ß1 as well as α2ß1. TC-I-15 was 100-fold more potent against α2ß1 binding to a lower-affinity collagen peptide, suggestive of a competitive mechanism. These results caution against the use of integrin inhibitors in a therapeutic or research setting without testing for cross-reactivity.

Integrin α2ß1 Targeting DGEA-Modified Liposomal Doxorubicin Enhances Antitumor Efficacy against Breast Cancer.

Breast cancer was the leading cause of newly diagnosed cases of tumors in 2020, ranking as the second highest cause of female death. Chemotherapy remains the conventional treatment of choice for breast tumors in most clinical cases. However, it is often accompanied by a poor prognosis and severe side effects, resulting from an insufficient accumulation of the drug at tumor sites and an unsystematic distribution of the drug across the body. Inspired by the fact that breast tumor cells overexpress integrin α2ß1 on the surface, we designed and constructed an integrin α2ß1 targeting DGEA-modified liposomal doxorubicin (DGEA-Lipo-DOX) platform for application in breast cancer therapy. The DGEA-Lipo-DOX was stable with a uniform particle size of 121.1 ± 3.8 nm and satisfactory drug encapsulation. Demonstrated in vitro and in vivo, the constructed platform exhibited improved antitumor ability. The DGEA-Lipo-DOX showed 4-fold enhanced blood circulation and 6-fold increased accumulation of DOX at the tumor sites compared to those of free DOX, resulting in a significantly enhanced antitumor efficacy in tumor-bearing mice. A preliminary safety evaluation suggested that the systemic toxicity of DOX was relieved by DGEA-Lipo delivery. Collectively, binding integrin α2ß1 by DGEA may represent an alternative therapeutic strategy for potentially safer breast cancer treatment.

RGD cadherins and α2ß1 integrin in cancer metastasis: A dangerous liaison.

We propose a new cadherin family classification comprising epithelial cadherins (cadherin 17 [CDH17], cadherin 16, VE-cadherin, cadherin 6 and cadherin 20) containing RGD motifs within their sequences. Expression of some RGD cadherins is associated with aggressive forms of cancer during the late stages of metastasis, and CDH17 and VE-cadherin have emerged as critical actors in cancer metastasis. After binding to α2ß1 integrin, these cadherins promote integrin ß1 activation, and thereby cell adhesion, invasion and proliferation, in liver and lung metastasis. Activation of α2ß1 integrin provokes an affinity increase for type IV collagen, a major component of the basement membrane and a critical partner for cell anchoring in liver and other metastatic organs. Activation of α2ß1 integrin by RGD motifs breaks an old paradigm of integrin classification and supports an important role of this integrin in cancer metastasis. Recently, synthetic peptides containing the RGD motif of CDH17 elicited highly specific and selective antibodies that block the ability of CDH17 RGD to activate α2ß1 integrin. These monoclonal antibodies inhibit metastatic colonization in orthotopic mouse models of liver and lung metastasis for colorectal cancer and melanoma, respectively. Hopefully, blocking the cadherin RGD ligand capacity will give us control over the integrin activity in solid tumors metastasis, paving the way for development of new agents of cancer treatment.

Integrin α2ß1-targeting ferritin nanocarrier traverses the blood-brain barrier for effective glioma chemotherapy.

BACKGROUND: Ferritin, the natural iron storage protein complex, self-assembles into a uniform cage-like structure. Human H-ferritin (HFn) has been shown to transverse the blood-brain barrier (BBB) by binding to transferrin receptor 1 (TfR1), which is abundant in endothelial cells and overexpressed in tumors, and enters cells via endocytosis. Ferritin is easily genetically modified with various functional molecules, justifying that it possesses great potential for development into a nanocarrier drug delivery system. RESULTS: In this study, a unique integrin α2ß1-targeting H-ferritin (2D-HFn)-based drug delivery system was developed that highlights the feasibility of receptor-mediated transcytosis (RMT) for glioma tumor treatment. The integrin targeting α2ß1 specificity was validated by biolayer interferometry in real time monitoring and followed by cell binding, chemo-drug encapsulation stability studies. Compared with naïve HFn, 2D-HFn dramatically elevated not only doxorubicin (DOX) drug loading capacity (up to 458 drug molecules/protein cage) but also tumor targeting capability after crossing BBB in an in vitro transcytosis assay (twofold) and an in vivo orthotopic glioma model. Most importantly, DOX-loaded 2D-HFn significantly suppressed subcutaneous and orthotopic U-87MG tumor progression; in particular, orthotopic glioma mice survived for more than 80 days. CONCLUSIONS: We believe that this versatile nanoparticle has established a proof-of-concept platform to enable more accurate brain tumor targeting and precision treatment arrangements. Additionally, this unique RMT based ferritin drug delivery technique would accelerate the clinical development of an innovative drug delivery strategy for central nervous system diseases with limited side effects in translational medicine.

Collagen I Modifies Connexin-43 Hemichannel Activity via Integrin α2ß1 Binding in TGFß1-Evoked Renal Tubular Epithelial Cells.

Chronic Kidney Disease (CKD) is associated with sustained inflammation and progressive fibrosis, changes that have been linked to altered connexin hemichannel-mediated release of adenosine triphosphate (ATP). Kidney fibrosis develops in response to increased deposition of extracellular matrix (ECM), and up-regulation of collagen I is an early marker of renal disease. With ECM remodeling known to promote a loss of epithelial stability, in the current study we used a clonal human kidney (HK2) model of proximal tubular epithelial cells to determine if collagen I modulates changes in cell function, via connexin-43 (Cx43) hemichannel ATP release. HK2 cells were cultured on collagen I and treated with the beta 1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFß1) ± the Cx43 mimetic Peptide 5 and/or an anti-integrin α2ß1 neutralizing antibody. Phase microscopy and immunocytochemistry observed changes in cell morphology and cytoskeletal reorganization, whilst immunoblotting and ELISA identified changes in protein expression and secretion. Carboxyfluorescein dye uptake and biosensing measured hemichannel activity and ATP release. A Cytoselect extracellular matrix adhesion assay assessed changes in cell-substrate interactions. Collagen I and TGFß1 synergistically evoked increased hemichannel activity and ATP release. This was paralleled by changes to markers of tubular injury, partly mediated by integrin α2ß1/integrin-like kinase signaling. The co-incubation of the hemichannel blocker Peptide 5, reduced collagen I/TGFß1 induced alterations and inhibited a positive feedforward loop between Cx43/ATP release/collagen I. This study highlights a role for collagen I in regulating connexin-mediated hemichannel activity through integrin α2ß1 signaling, ahead of establishing Peptide 5 as a potential intervention.

Biphasic α2ß1 Integrin Expression in Breast Cancer Metastasis to Bone.

Integrins participate in the pathogenesis and progression of tumors at many stages during the metastatic cascade. However, current evidence for the role of integrins in breast cancer progression is contradictory and seems to be dependent on tumor stage, differentiation status, and microenvironmental influences. While some studies suggest that loss of α2ß1 enhances cancer metastasis, other studies suggest that this integrin is pro-tumorigenic. However, few studies have looked at α2ß1 in the context of bone metastasis. In this study, we aimed to understand the role of α2ß1 integrin in breast cancer metastasis to bone. To address this, we utilized in vivo models of breast cancer metastasis to bone using MDA-MB-231 cells transfected with an α2 expression plasmid (MDA-OEα2). MDA cells overexpressing the α2 integrin subunit had increased primary tumor growth and dissemination to bone but had no change in tumor establishment and bone destruction. Further in vitro analysis revealed that tumors in the bone have decreased α2ß1 expression and increased osteolytic signaling compared to primary tumors. Taken together, these data suggest an inverse correlation between α2ß1 expression and bone-metastatic potential. Inhibiting α2ß1 expression may be beneficial to limit the expansion of primary tumors but could be harmful once tumors have established in bone.

Interactions via α2ß1 Cell Integrin May Protect against the Progression of Airway Structural Changes in Asthma.

Increased airway wall thickness and remodeling of bronchial mucosa are characteristic of asthma and may arise from altered integrin signaling on airway cells. Here, we analyzed the expression of ß1-subfamily integrins on blood and airway cells (flow cytometry), inflammatory biomarkers in serum and bronchoalveolar lavage, reticular basement membrane (RBM) thickness and collagen deposits in the mucosa (histology), and airway geometry (CT-imaging) in 92 asthma patients (persistent airflow limitation subtype: n = 47) and 36 controls. Persistent airflow limitation was associated with type-2 inflammation, elevated soluble α2 integrin chain, and changes in the bronchial wall geometry. Both subtypes of asthma showed thicker RBM than control, but collagen deposition and epithelial α1 and α2 integrins staining were similar. Type-I collagen accumulation and RBM thickness were inversely related to the epithelial expression of the α2 integrin chain. Expression of α2ß1 integrin on T-cells and eosinophils was not altered in asthma. Collagen I deposits were, however, more abundant in patients with lower α2ß1 integrin on blood and airway CD8+ T-cells. Thicker airway walls in CT were associated with lower α2 integrin chain on blood CD4+ T-cells and airway eosinophils. Our data suggest that α2ß1 integrin on inflammatory and epithelial cells may protect against airway remodeling advancement in asthma.

Type I Collagen Signaling Regulates Opposing Fibrotic Pathways through α2ß1 Integrin.

Fibrosis is characterized by fibroblast activation, leading to matrix remodeling culminating in a stiff, type I collagen-rich fibrotic matrix. Alveolar epithelial cell (AEC) apoptosis is also a major feature of fibrogenesis, and AEC apoptosis is sufficient to initiate a robust lung fibrotic response. TGF-ß (transforming growth factor-ß) is a major driver of fibrosis and can induce both AEC apoptosis and fibroblast activation. We and others have previously shown that changes in extracellular matrix stiffness and composition can regulate the cellular response to TGF-ß. In the present study, we find that type I collagen signaling promotes TGF-ß-mediated fibroblast activation and inhibits TGF-ß-induced AEC death. Fibroblasts cultured on type I collagen or fibrotic decellularized lung matrix had augmented activation in response to TGF-ß, whereas AECs on cultured on type I collagen or fibrotic lung matrix were more resistant to TGF-ß-induced apoptosis. Both of these responses were mediated by integrin α2ß1, a major collagen receptor. AECs treated with an α2 integrin inhibitor or with deletion of α2 integrin had loss of collagen-mediated protection from apoptosis. We found that mice with fibroblast-specific deletion of α2 integrin were protected from fibrosis whereas mice with AEC-specific deletion of α2 integrin had more lung injury and a greater fibrotic response to bleomycin. Intrapulmonary delivery of an α2 integrin-activating collagen peptide inhibited AEC apoptosis in vitro and in vivo and attenuated the fibrotic response. These studies underscore the need for a thorough understanding of the divergent response to matrix signaling.

The stiffness-controlled release of interleukin-6 by cardiac fibroblasts is dependent on integrin α2ß1.

Cardiac fibroblasts are able to sense the rigidity of their environment. The present study examines whether the stiffness of the substrate in cardiac fibroblast culture can influence the release of interleukin-6 (IL-6), interleukin-11 (IL-11) and soluble receptor of IL-6 (sIL-6R). It also examines the roles of integrin α2ß1 activation and intracellular signalling in these processes. Cardiac fibroblasts were cultured on polyacrylamide gels and grafted to collagen, with an elasticity of E = 2.23 ± 0.8 kPa (soft gel) and E = 8.28 ± 1.06 kPa (stiff gel, measured by Atomic Force Microscope). Flow cytometry and ELISA demonstrated that the fibroblasts cultured on the soft gel demonstrated higher expression of the α2 integrin subunit and increased α2ß1 integrin count and released higher levels of IL-6 and sIL-6R than those on the stiff gel. Substrate elasticity did not modify fibroblast IL-11 content. The silencing of the α2 integrin subunit decreased the release of IL-6. Similar effects were induced by TC-I 15 (an α2ß1 integrin inhibitor). The IL-6 levels in the serum and heart were markedly lower in α2 integrin-deficient mice B6.Cg-Itga2tm1.1Tkun/tm1.1Tkun than wild type. Inhibition of Src kinase by AZM 475271 modifies the IL-6 level. sIL-6R secretion is not dependent on α2ß1 integrin. Conclusion: The elastic properties of the substrate influence the release of IL-6 by cardiac fibroblasts, and this effect is dependent on α2ß1 integrin and kinase Src activation.